This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e. proteotypic peptides). To develop a catalog of proteotypic peptides in human heart, TRIzol? extracts of left-ventricular tissue from non-failing and failing human heart explants were optimized for shotgun proteomic analysis using Multi-dimensional Protein Identification Technology (MudPIT). Ten replicate MudPIT analyses were performed on each tissue sample and resulted in the identification 30,605 unique peptides with a q-value of 0.01 or better, corresponding to 7,138 unique human heart proteins. Experimental observation frequencies were assessed and used to select over 4,476 proteotypic peptides for 2,558 heart proteins. This human cardiac dataset can serve as a public reference to guide the selection of proteotypic peptides for future targeted mass spectrometry experiments monitoring potential protein biomarkers of human heart diseases.